The Therapeutic Potential of Cold Stored Platelets in Regulating Vascular Stability in Trauma Trauma is the leading cause of death world-wide in individuals between the ages of 1-44, with traumatic brain injury (TBI) being the number one cause of death after trauma. Platelet transfusion and balanced ratios of blood products have been shown to increase survival in severely injured bleeding trauma patients. In the current US blood-banking practice, platelets (Plts) are stored in incubators at 22C for up to 5 days. Storage of Plts at 22C for 5 days is associated with a storage lesion, increased infectious risk, and an overall decline in hemostatic function. 4C storage of Plts has been proposed as an equivalent and in some cases superior alternative to 22 C storage. Therapeutically, in addition to their critical role in hemostasis, Plts are known to safeguard the integrity of the vascular endothelium. Vascular instability is a hallmark effect of traumatic injury leading to vascular permeability, inflammation, coagulation disturbances and end organ failure. In TBI, intracranial hemorrhage (ICH) and cerebral edema are the leading causes of mortality, which are potentially addressable by Plt transfusion. Our previous data demonstrate that 4C Plts regulate vascular stability and inhibit endothelial cell (EC) permeability similar to 22C Plts. This proposal will aim to elucidate the therapeutic effects of platelets and cold stored (4oC) platelets on vascular stability in traumatic brain injury (TBI) with the thought that platelets can be used as a first line therapeutic intervention in TBI to decrease cerebral edema, ICH, neuroinflammation and improve outcomes in TBI patients. Aim 1 is a mechanistic aim designed to investigate the structural, molecular and cellular signaling effects of 4C storage on Plts and on Plt-endothelial cell interactions and coagulation. Inhibitors of relevant Plt and EC signaling pathways will be utilized to tease apart what pathways are relevant to Plt effects on endothelial permeability. Aim 2 is designed to provide a deeper understanding of the effects of 4C Plts on the vascular endothelium by using state of the art intravital imaging in the cremaster muscle and brain, to visualize the effects of 4C Plts on vascular permeability, clot formation and preservation of the endothelial glycocalyx. Aim 3 is designed to test out our primary hypothesis that 4C Plts can be used to regulate vascular stability in TBI hence improving hemostasis, decreasing neuroinflammation and neuronal cell loss. In this revised proposal we have demonstrated our full capabilitiy to execute the proposed studies and have refined our experiments to successfully answer the questions posed.